Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium

TLDR

The study evaluated a microplate Alamar blue assay for rapid, inexpensive high‑throughput antimycobacterial drug screening. MICs of 30 antimicrobials were measured in MABA using visual and fluorometric readouts and compared to BACTEC 460 results. MABA produced MICs within one dilution of BACTEC for most drugs, showed high correlation with BACTEC and between visual and fluorometric readouts, and proved sensitive, rapid, inexpensive, and non‑radiometric, making it suitable for large‑scale screening and indicating H37Ra as a surrogate for H37Rv.

Abstract

In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.